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Biomedical subjects

R Narayanan

Publications and source records attributed to R Narayanan.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synaptic localization and restricted diffusion of a Drosophila neuronal synaptobrevin--green fluorescent protein chimera in vivo.

Fluorescent markers for subcellular compartments in Drosophila neurons should allow one to combine genetic mutant analysis with visualization of subcellular structures in vivo. Here we describe an analysis of two markers which may be used to observe different compartments of live Drosophila synapses. Soluble jellyfish green fluorescent protein (GFP) expressed at high levels in neurons diffuses freely in the neuronal cytosol as evidenced by confocal microscopy and fluorescence recovery from photobleaching experiments. Thus, the distribution pattern of soluble GFP in motor axons and larval motor terminals indicates the expected distribution for diffusible presynaptic molecules. In contrast to GFP, a neurally expressed neuronal synaptobrevin-GFP chimera (n-syb GFP) is transported down axons and specifically localized to nerve terminals. We demonstrate that n-syb GFP labels synaptic-vesicle membrane at larval motor terminals by documenting its restriction to presynaptic varicosities, its colocalization with synaptic vesicle antigens, and its redistribution in Drosophila shits1 mutant nerve terminals transiently depleted of synaptic vesicles. Surprisingly, n-syb GFP expressed in muscle is concentrated at the subsynaptic reticulum (SSR), postsynaptic infoldings of muscle plasma membrane. We suggest, using different membrane markers, that this apparent postsynaptic enrichment simply reflects a concentration of plasma membrane in the SSR, rather than a selective targeting of n-syb GFP to postsynaptic sites. Utilities and implications of these studies are demonstrated or discussed.

Animals↗
PubMed1998

Telomeric DNA: marker for human prostate cancer development?

BACKGROUND: Telomeres that protect chromosomes at both ends are shortened with each somatic cell division through replication-dependent sequence loss at DNA termini. The chromosomes with shortened telomeres tend to become unstable, leading to cell death. Due largely to reactivation/upregulation of telomerase, a ribonucleoprotein that adds nucleotide sequences onto chromosome ends, cancer cells become immortal and neoplastically transformed. METHODS: The purpose of the present study was to study three newly established human prostate cancer cell lines and three prostate-derived fibroblastic cell cultures at different passages for telomeric DNA signal intensity, telomeric restriction fragment length (TRFL), telomerase activity, and spontaneous apoptotic index. RESULTS: Compared with the three fibroblastic cell cultures, the three new prostate cancer cell lines showed: 1) telomerase activity, 2) stronger telomeric signals, 3) relatively longer TRFLs, and 4) much lower apoptotic indices. On the other hand, three fibroblastic cell cultures showed: 1) no telomerase activity, 2) weaker telomeric signals, 3) shorter TRFLs (fibroblasts derived from surrounding tissue of prostate tumor showed intermediate TRFLs), and 4) comparatively higher apoptotic indices. CONCLUSIONS: Based on these results, we conclude that telomeric DNA signal intensity, TRFL, and telomerase activity can be used to distinguish prostate cancer cells from adjacent fibroblasts.

Apoptosis↗
PubMed1998

Enhancement by antisense oligonucleotides to NF-kappaB of the differentiation of HL-60 promyelocytic leukemia cells induced by vitamin D3.

We have demonstrated previously that a phosphorothioate antisense oligonucleotide to the p65 subunit of the inducible transcription factor NF-kappaB produced rapid changes in the expression of leukocyte integrin CD11b (Mo 1) and in the adhesion of dimethylsulfoxide (DMSO)-differentiated HL-60 cells stimulated by 12-O-tetradecanoylphorbol 13-acetate. We have also shown that a variety of agents which inhibit NF-kappaB, including vitamin E and related antioxidants, curcumin and several non-steroidal anti-inflammatory agents, significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of 1,25-dihydroxyvitamin D3 (vitamin D3). To provide further evidence that interference with the activation of NF-kappaB affects the maturation of HL-60 leukemia cells by creating an environment conducive to terminal differentiation, we measured the effects of phosphorothioate antisense oligonucleotides to the various subunits of NF-kappaB on the differentiation of HL-60 cells produced by low levels of vitamin D3. When used alone these oligonucleotides had no significant effect on the differentiation of HL-60 cells. However, the antisense oligomer to the Rel A subunit of NF-kappaB markedly increased the extent of differentiation produced by low levels of vitamin D3. An enhancement of the differentiation of HL-60 cells induced by vitamin D3 was also obtained by several transcription factor decoys designed to mimic the consensus sequences of genes activated by Rel A. The findings provide additional support for the concept that inhibition of the activation of NF-kappaB may be involved in regulating the entry of promyelocytic leukemia cells into a differentiation pathway.

Cell Differentiation↗
PubMed1998

Expression profile of the putative catalytic subunit of the telomerase gene.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called telomeres to the growing ends of chromosomal DNA. The enzyme telomerase activity is present in a vast majority of tumors but is repressed in most normal tissues. Recently, two groups have reported the molecular cloning of the putative catalytic subunit (hEST2/hTRT) of the telomerase gene. We investigated the expression of this gene in diverse tumor-derived cell lines and tumors as well as in various normal tissues. The expression of hEST2/hTRT was detectable in tumor-derived cell lines, primary breast tumors, pancreatic tumors, and kidney tumors. Furthermore, the expression of hEST2/hTRT was down-regulated in response to a differentiation inducer. However, several normal tissues also expressed varying levels of hEST2/hTRT. Early passage cultures of endothelial fibroblasts and some epithelial cells also expressed the telomerase gene, albeit at low levels. In contrast, the expression of TLP1/TP1, the human homologue of Tetrahymena p80 telomerase subunit, was similar in all of these samples. Our results indicate that the differences in expression of hEST2/hTRT in tumor versus normal cells are relative and are not absolute.

Breast Neoplasms↗
PubMed1997

Characterization of human telomerase complex.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called "telomeres" to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

Amino Acid Sequence↗
PubMed1996

Cell cycle-dependent modulation of telomerase activity in tumor cells.

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The highest level of telomerase activity was detected in S-phase cells. In contrast, cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. Diverse cell cycle blockers, including transforming growth factor beta1 and cytotoxic agents, also caused inhibition of telomerase activity. These results establish a direct link between telomerase activity and progression through the cell cycle.

Antineoplastic Agents↗
PubMed1996

Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection.

Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.

Cells, Cultured↗
PubMed1996

Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides.

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.

3T3 Cells↗
PubMed1996

Telomerase as a potential molecular target to study G-quartet phosphorothioates.

Inhibition of gene expression by phosphorothioate oligomers is complex and involves specific and nonspecific mechanisms. Oligomers that contain a G-quartet elicit distinct effects in vitro and in vivo that are dependent on the context of the G-quartet's occurrence within a sequence. The enzyme telomerase, a ribonucleoprotein, has a stretch of C residues in the RNA template, which are used to add terminal dG-rich telomeric repeats to the ends of chromosomes. Some but not all phosphorothioates containing a G-quartet, depending on the context of occurrence, inhibited telomerase activity in vitro. Non-G-quartet phosphorothioates did not inhibit this activity. Activities of control enzymes, such as reverse transcriptase or taq polymerase, were not affected by the G-quartet oligomers. Neither phosphodiester nor chimeric oligomers of a G-quartet-containing oligomer were as potent inhibition of telomerase activity as phosphorothioate oligomers. These results may provide a molecular target to study the effects of G-quartet-containing oligomers.

Animals↗
PubMed1996

Antisense therapy.

The sequence specificity of the antisense technique makes it an attractive basis for novel molecular therapeutics. Inhibition of gene expression by antisense in cell culture models has provided a strong rationale for identification and validation of disease targets. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials of diseases including AIDS, cancer, and inflammation. It is becoming increasingly apparent that these drugs act by means of a complex mechanism of action, and some of their effects can be sequence independent. Nevertheless, these oligodeoxynucleotides offer considerable promise as novel molecular drugs. Harnessing the therapeutic potential of this powerful technique depends on elucidation of the complex mechanism of action so that effective and meaningful therapeutic modalities can be realized.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Increased quinolinate immunoreactivity in the peripheral blood monocytes/macrophages from SIV-infected monkeys.

Quinolinate (QUIN), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that is thought to act through the NMDA receptor system, was localized in cultured peripheral blood monocytes/macrophages from SIV-infected monkeys using a recently developed immunohistochemical method. Significant increases in QUIN immunoreactive (IR) cells were detected in all five SIV-infected monkeys examined. Multinucleated giant cells, a hallmark of lentiviral infection, were visible in selected samples. Treatment with the QUIN precursors, tryptophan and kynurenine, increased the number of QUIN-IR cells in both the control and SIV-infected preparations, perhaps by a mass action mechanism. We hypothesize that in SIV-infected monkeys, infiltrating monocytes/macrophages contribute to the high level of brain QUIN and associated neuropathology.

Animals↗
PubMed1996

Stromal cells regulate bcl-2 and bax expression in pro-B cells.

B lymphocyte production in the bone marrow depends on a cascade of regulatory cells and cytokines unique to the hematopoietic microenvironment. Fibroblastic stromal cells appear to be particularly important in regulating the earliest events in this lineage; however, it is still not clear whether the same or different sets of signals regulate maintenance of cell viability, proliferation, and differentiation of B lineage cells. In this study, we addressed the role of bone marrow stromal cells in survival and expansion of normal murine pro-B cells. Stromal cells were required for long-term proliferation of pro-B cell clone C1.92, and, in the presence of stromal cell line S10, pro-B cells expressed the proto-oncogene bcl-2. Removal of C1.92 cells from Stromal cell-derived signaling in support of pro-B cell viability. Due to its previously described role in regulating cell survival, we investigated whether stromal cells regulate bcl-2 expression in pro-B cells. When removed from stromal cell cultures, pro-B cells rapidly lost bcl-2 mRNA expression coincident with initiation of apoptosis. However, interruption of bcl-2 expression with antisense oligonucleotides in the presence of stroma and interleukin-7 (IL-7) did not result in immediate cell death. Oligonucleotide-treated cells arrested in G(1) phase of the cell cycle 24 hours before the initiation of apoptosis. In contrast, removal of pro-B cells from stromal cell support resulted in rapid increase in BAX expression, correlating directly with initiation of apoptosis. These results suggest that bcl-2 may, in part, regulate cell survival by interrupting the cascade of intracellular events that regulate cell cycle progression in lymphopoietic cells. Initiation of apoptosis in these cells appears to be more closely correlated with intracellular levels of BAX expression.

Animals↗
PubMed1995

Differentiation of immortal cells inhibits telomerase activity.

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Animals↗
PubMed1995

Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

Animals↗